Automated immunohistochemical and in situ hybridization assay formulations

ABSTRACT

The present invention provides reagents for use in an automated environment for cell conditioning of biological samples wherein the cells or tissues are predisposed for access by reagent molecules for histochemical and cytochemical staining procedures. The reagents comprise components optimized to faciliate molecular access to cells and cell constituents within the biological sample. The present invention also provides reagents for use in an automated environment for removing or etching embedding media by exposing a biological sample to be stained in histochemical or cytochemical procedures without the dependence on organic solvents. The reagents comprise components optimized to facilitate removal or etching of the embedding media from the biological sample.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/800,689, filed Mar. 7, 2001, which is a continuation-in-part of U.S.application Ser. No. 09/721,096, filed Nov. 22, 2000, with Atty. DocketNo. 98,541-C. U.S. application Ser. No. 09/721,096 claimed the benefitof priority from International Application No. PCT/US99/20353, filedSep. 3, 1999, which was published on Mar. 16, 2000 as WO 00/14507.International Application No. PCT/US99/20353, in turn, claimed thebenefit of priority from U.S. Provisional Patent Application No.60/099,018, filed Sep. 3, 1998; U.S. patent application Ser. No.09/259,240, filed Feb. 26, 1999; and International Application No.PCT/US99/04181, filed Feb. 26, 1999, which was published on Sep. 2, 1999as WO 99/44030. U.S. patent application Ser. No. 09/721,096, filed Nov.22, 2000; International Patent App. No. PCT/US99/20353, filed Sep. 3,1999, and published as WO 00/14507 on Mar. 16, 2000; U.S. ProvisionalApp. No. 60/099,018, filed Sep. 3, 1998; U.S. patent application Ser.No. 09/259,240, filed Feb. 26, 1999 and International Patent App. No.PCT/US99/04181, filed Feb. 26, 1999, and published as WO 99/44030 onSep. 2, 1999, are all hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to solutions methods, and instruments forconditioning cells or tissues so as to increase the accessibility ofvarious molecules to their respective targets and generally to improvetissue and cell readability of biological samples on automatedinstruments prior to immunohistochemical (IHC), in situ hybridization(ISH) or other histochemical or cytochemical manipulations.

2. Summary of the Related Art

All patents, patent applications and non-patent articles or referencesmentioned herein are hereby incorporated by reference to the extent thatthey are not contradictory.

The diagnosis of disease based on the interpretation of tissue or cellsamples taken from a diseased organism has expanded dramatically overthe past few years. In addition to traditional histological stainingtechniques and immunohistochemical assays, ill situ techniques such asin situ hybridization and in situ polymerase chain reaction are now usedto help diagnose disease states in humans. Thus, there are a variety oftechniques that can assess not only cell morphology, but also thepresence of specific macromolecules within cells and tissues.

For example, the diagnosis of breast, ovarian and other carcinomas maybe facilitated by the use of techniques designed to identify thepresence or absence of the c-erb2/HER-2/neu protooncogene or theprotein(s) expressed therefrom. The c-erb2/HER-2/neu protooncogene is amember of the epidermal growth factor receptor (EGFR) family of receptortyrosine kinases. Amplification and overexpression of thec-erb2/HER-2/neu protooncogene is found in about 30% of breastcarcinomas, about 20% of ovarian carcinomas and others. (Andrechek, E.R. et al., Proc. Natl. Acad. Sci. USA, vol. 97, no. 7, pp. 3444-3449(2000); Doherty, J. K. et al., Proc. Natl. Acad. Sci. USA, vol. 96, pp.10869-10874 (1999); Oh, J. J. et al., Nucleic Acids Res., vol. 27, no.20, pp. 4008-4017 (1999); Klapper, L. N. et al., Proc. Natl. Acad. Sci.USA, vol. 96, pp. 4995-5000 (1999); and references found in each of theaforementioned articles.)

Each of these techniques requires that sample cells or tissues undergopreparatory procedures that may include fixing the sample with chemicalssuch as an aldehyde (such as formaldehyde, glutaraldehyde), formalinsubstitutes, alcohol (such as ethanol, methanol, isopropanol) orembedding the sample in inert materials such as paraffin, celloidin,agars, polymers, resins, cryogenic media or a variety of plasticembedding media (such as epoxy resins and acrylics). Other sample tissueor cell preparations require physical manipulation such as freezing(frozen tissue section) or aspiration through a fine needle (fine needleaspiration (FNA)). Regardless of the tissue or cell sample or its methodof preparation or preservation, the goal of the technologist is toobtain accurate, readable and reproducible results that permit theaccurate interpretation of the data. One way to provide accurate,readable and reproducible data is to prepare the tissue or cells in afashion that optimizes the results of the test regardless of thetechnique employed. In the case of immunohistochemistry and in situtechniques this means increasing the amount of signal obtained from thespecific probe (e.g. antibody, DNA, RNA, etc.). In the case ofhistochemical staining it may mean increasing the intensity of the stainor increasing staining contrast.

Without preservation, tissue samples rapidly deteriorate such that theiruse in diagnostics is compromised shortly after removal from their host.In 1893, Ferdinand Blum discovered that formaldehyde could be used topreserve or fix tissue so that it could be used in histochemicalprocedures. The exact mechanisms by which formaldehyde acts in fixingtissues are not fully established, but they involve cross-linking ofreactive sites within the same protein and between different proteinsvia methylene bridges (Fox et al., J. Histochem. Cytochem. 33: 845-853(1985)). Recent evidence suggests that calcium ions also play a role(Morgan et al., J. Path. 174: 301-307 (1994)). These links cause changesin the quaternary and tertiary structures of proteins, but the primaryand secondary structures appear to be preserved (Mason et al., J.Histochem. Cytochem. 39: 225-229 (1991)). The extent to which thecross-linking reaction occurs depends on conditions such as theconcentration of formalin, pH, temperature and length of fixation (Foxet al., J. Histochem. Cytochem. 33: 845-853 (1985)). Some antigens, suchas gastrin, somatostatin and α-1-antitrypsin, may be detected afterformalin fixation, but for many antigens, such as intermediate filamentsand leukocyte markers, immunodetection after formalin treatment is lostor markedly reduced (McNicol & Richmond, Histopathology 32: 97-103(1998)). Loss of antigen immunoreactivity is most noticeable at antigenepitopes that are discontinuous, i.e. amino acid sequences where theformation of the epitope depends on the confluence of portions of theprotein sequence that are not contiguous.

Antigen retrieval refers to the attempt to “undo” the structural changesthat treatment of tissue with a cross-linking agent induces in theantigens resident within that tissue. Although there are severaltheories that attempt to describe the mechanism of antigen retrieval(e.g., loosening or breaking of crosslinkages formed by formalinfixation), it is clear that modification of protein structure byformalin is reversible under conditions such as high-temperatureheating. It is also clear that several factors affect antigen retrieval:heating, pH, molarity and metal ions in solution (Shi et al., J.Histochem. Cytochem. 45: 327-343 (1997)).

Microwave heating appears to be the most important factor for retrievalof antigens masked by formalin fixation. Microwave heating (100°±5° C.)generally yields better results in antigen retrievalimmunohistochemistry (AR-IHC).

Different heating methods have been described for antigen retrieval inIHC such as autoclaving (Pons et al, Appl. Immunohistochem. 3: 265-267(1995); Bankfalvi et al., J. Path. 174: 223-228 (1994)); pressurecooking (Miller & Estran, Appl. Immunohistochem. 3: 190-193 (1995);Norton et al., J. Path. 173: 371-379 (1994)); water bath (Kawai et al.,Path. Int. 44: 759-764 (1994)); microwaving plus plastic pressurecooking (U.S. Pat. No. 5,244,787; Pertschuk et al., J. Cell Biochem.19(suppl.): 134-137 (1994)); and steam heating (Pasha et al., Lab.Invest. 72: 167A (1995); Taylor et al., CAP Today 9: 16-22 (1995)).

Although some antigens yield satisfactory results when microwave heatingis performed in distilled water, many antigens require the use ofbuffers during the heating process. Some antigens have particular pHrequirements such that adequate results will only be achieved in anarrow pH range. Presently, most antigen retrieval solutions are used ata pH of approximately 6-8, but there is some indication that slightlymore basic solutions may provide marginally superior results (Shi, etal., J. Histochem. Cytochem. 45: 327-343 (1997)).

Although the chemical components of the antigen retrieval solution,including metal ions, may play a role as possible co-factors in themicrowave heating procedure, thus far, no single chemical has beenidentified that is both essential and best for antigen retrieval.

Many solutions and methods are used routinely for staining enhancements.These may include but are not limited to distilled water, EDTA, urea,Tris, glycine, saline and citrate buffer. Solutions containing a varietyof detergents (ionic or non-ionic surfactants) may also facilitatestaining enhancement under a wide range of temperatures (from ambient toin excess of 100° C.).

In addition to cell surface molecules that may be present on theexterior portion of the cell, other molecules of interest in IHC, ISHand other histochemical and cytochemical manipulations are locatedwithin the cell, often on the nuclear envelope. Some of these moleculesundergo molecular transformation when exposed to a fixative (coagulativeor precipitive) such as formalin. Thus with respect to these moleculesit is desirable to not only overcome the effects of fixation but also toincrease the permeability of the cell in order to facilitate theinteraction of organic and inorganic compounds with the cell.

Other tissue samples may not have been subjected to cross-linking agentsprior to testing, but improved results with respect to these tissues isalso important. There are a variety of non-formalin methods forpreserving and preparing cytological and histological samples. Examplesof these methods include, but are not limited to: a) hematology smears,cytospins™, ThiinPreps™, touch preps, cell lines, Ficoll separations,etc. are routinely preserved in many ways which includes but are notlimited to, air-drying, alcoholic fixation, spray fixatives and storagemediums such as sucrose/glycerin; b) tissues and cells (either fixed orunfixed) may be frozen and subsequently subjected to various stabilizingtechniques which include, but are not limited to, preservation, fixationand desiccation; c) tissues and cells may be stabilized in a number ofnon-cross-linking aldehyde fixatives, non-aldehyde containing fixatives,alcoholic fixatives, oxidizing agents, heavy metal fixatives, organicacids and transport media.

One way to improve testing results is to increase the signal obtainedfrom a given sample. In a general sense, increased signal can beobtained by increasing the accessibility of a given molecule for itstarget. As in the case for antigens found within the cell, targetswithin the cell can be made more accessible by increasing thepermeability of the cell thereby permitting a greater number ofmolecules entry into the cell, thereby increasing the probability thatthe molecule will “find” its target. Such increased permeability isespecially important for techniques such as ISH, in situ PCR, IHC,histochemistry and cytochemistry.

Tissues and cells are also embedded in a variety of inert media(paraffin, celloidin, OCT™, agar, plastics or acrylics etc.) to helppreserve them for future analysis. Many of these inert materials arehydrophobic and the reagents used for histological and cytologicalapplications are predominantly hydrophilic; therefore, the inert mediummay need to be removed from the biological sample prior to testing. Forexample, paraffin embedded tissues sections are prepared for subsequenttesting by removal of the paraffin from the tissue section by passingthe slide through various organic solvents such as toluene, xylene,limonene or other suitable solvents. These organic solvents are veryvolatile causing a variety of problems including requiring specialprocessing (e.g., deparaffinization is performed in ventilated hoods)and requires special waste disposal. The use of these organic solventsincreases the cost of analysis and exposure risk associated with eachtissue sample tested and has serious negative effects for theenvironment.

Presently, there is no available technique for removing inert media fromsample tissue by directly heating the slide in an automated fashion.Neither is it currently possible to remove inert media from sampletissue while conditioning the sample tissue or cell in a one-stepautomated staining process.

The methods of the present invention permit a) automated removal ofembedding media without the use of organic solvents, thus exposing thecells for staining and thereby reducing time, cost and safety hazards,b) automated cell conditioning without automated removal of embeddingmedia from the sample cell or tissue, c) a multi-step automated processthat exposes the cells, performs cell conditioning and increasespermeability of the cytological or histological specimens, therebyincreasing sample readability and improving interpretation of test data.The methods of the present invention can be used for improving thestainability and readability of most histological and cytologicalsamples used in conjunction with cytological and histological stainingtechniques.

SUMMARY OF THE INVENTION

The present invention relates to solutions, methods and instruments forcell or tissue conditioning, which improves the accessibility ofmolecules in biological samples during histochemical or cytochemicalassays. The conditioning may be performed at any point in ahistochemical or cytochemical protocol.

The present invention further relates to an automated method forexposing biological samples for use in histological or cytologicaltesting procedures by removing the embedding media without the use oforganic solvents.

The present invention also relates to an automated method for thesimultaneous exposing and cell conditioning of biological samples forhistochemical and cytochemical applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an instrument in which the formulationsof the present invention may be used shown with the slide hood open andthe carousel door removed.

FIG. 2 is a perspective view of an instrument in which the formulationsof the present invention may be used shown in conjunction with acomputer and other instruments with which it operates.

FIG. 3 is an exploded view of an instrument in which the formulations ofthe present invention may be used.

FIG. 4 is a perspective view of an instrument in which the formulationsof the present invention may be used shown with a reagent dispenser.

FIG. 5 is a block diagram of the heating system of an instrument inwhich the formulations of the present invention may be used.

FIG. 6 is a flow chart for one embodiment for removing the embeddingmedia from a slide.

FIGS. 7 a-b are flow charts for one embodiment for cell conditioning.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides reagents, formulations, aqueouscompositions of matter and the like for use in an automated environmentfor cell conditioning of biological samples wherein the cells or tissuesare predisposed for access by reagent molecules for histochemical andcytochemical staining procedures. The reagents comprise componentsoptimized to facilitate molecular access to cells and cell constituentswithin the biological sample. The present invention also providesreagents for use in an automated environment for removing or etchingembedding media by exposing a biological sample to be stained inhistochemical or cytochemical procedures without the dependence onorganic solvents. The reagents comprise components optimized tofacilitate removal or etching of the embedding media from the biologicalsample.

One embodiment in which the formulations of the present invention may beuseful relates to the exposing of biological samples by removal of theinert materials in which biological samples have been embedded forpreservation and support. In a preferred embodiment, paraffin or otherinert materials are removed from biological samples by heating one sideof the biological sample. This may be accomplished by contact heating ofthe microscope slide on which the embedded biological samples have beenplaced. Other inert materials that can be removed from embeddedbiological samples include, but are not limited to, agars and cryogenicmedia. This process of removal of inert embedding media or etching ofembedding media is referred to herein as exposing.

In a preferred method in which the formulations of the present inventionmay be useful, the paraffin-embedded biological sample laying on theglass slide is first heated by a heating element. The heating elementexposes heat on one side of the biological sample (such as the thermalplatforms disclosed in U.S. patent application Ser. No. 09/259,240,herein incorporated by reference) within an automated staininginstrument (U.S. patent application Ser. No. 08/995,052 filed on Dec.19, 1997 and U.S. provisional patent application Ser. No. 60/076,198filed on Feb. 27, 1998, both of which are herein incorporated byreference) such that the sample slide is dried and the paraffin ismelted.

Heating Elements

As discussed in U.S. patent application Ser. No. 09/259,240 (which isincorporated by reference) and referring now in detail to the drawingswherein like parts are designated by like reference numerals throughout,there is illustrated in FIG. 1 a perspective view of the molecularpathology apparatus in which formulations according to the presentinvention are useful, such apparatus is designated generally byreference numeral 10. Apparatus 10 is designed to automatically stain orotherwise treat tissue mounted on microscope slides with nucleic acidprobes, antibodies, and/or reagents associated therewith in the desiredsequence, time and temperature. Tissue sections so stained or treatedare then to be viewed under a microscope by a medical practitioner whoreads the slide for purposes of patient diagnosis, prognosis, ortreatment selection.

In a preferred embodiment of an apparatus in which the formulations ofthe present invention are useful, apparatus 10 functions as onecomponent or module of a system 12 (FIG. 2), which also comprises a hostcomputer 14 preferably a personal computer, monitor 16, keyboard 18,mouse 20, bulk fluid containers 22, waste container 23 and relatedequipment. Additional staining modules or other instruments may be addedto system 12 to form a network with computer 14 functioning as a server.Alternatively, some or all of these separate components could beincorporated into apparatus 10 making it a stand-alone instrument.

The preferred configuration of apparatus 10 as well as system 12 isgenerally as described in U.S. patent application Ser. No. 08/995,052filed Dec. 19, 1997 as well as in the Ventana NexES User's Guideavailable from Ventana Medical Systems, Inc. (Tuscon, Ariz.), bothincorporated herein by reference, except with respect to the novelheating system, slide support, bulk fluids module, volume adjust, andslide wipe as disclosed below. For purposes of clarity, detaileddescriptions of those components found in both the present invention andthe incorporated references are omitted.

In brief, apparatus 10 is a microprocessor controlled staininginstrument that automatically applies chemical and biological reagentsto tissue mounted on standard glass microscope slides. A carouselsupporting radially positioned glass slides is revolved by a steppermotor to place each slide under one of a series of reagent dispensers.Apparatus 10 controls dispensing, washing, mixing, and heating tooptimize reaction kinetics. The computer controlled automation permitsuse of apparatus 10 in a walk-away manner, i.e. with little manuallabor.

More particularly, apparatus 10 comprises a housing formed of a lowersection 30 removably mounted or hinged to an upper section 32. A slidecarousel 34 is mounted within lower section 30 for rotation about axisA-A. As set forth in greater detail below, a plurality of thermalplatforms 50 are mounted radially about the perimeter of carousel 34upon which standard glass slides with tissue samples may be placed.Carousel 34 is preferably constructed of stainless steel. It is a keyfeature of the present invention that the temperature of each slide maybe individually controlled by means of various sensors andmicroprocessors as described herein. Also housed within apparatus 10(FIG. 3) are wash dispense nozzles 36, Coverslip™ dispense nozzle 37,fluid knife 38, wash volume adjust nozzle 39, bar code reader mirror 40,and air vortex mixers 42 the details of which are discussed hereinafter.

Rotatably mounted atop upper section 32 is a reagent carousel 28.Dispensers 26 are removably mounted to reagent tray 29 (FIG. 4) which,in turn, is adapted to engage carousel 28. Reagents may include anychemical or biological material conventionally applied to slidesincluding nucleic acid probes or primers, polymerase, primary andsecondary antibodies, digestion enzymes, pre-fixatives, post-fixatives,blocking agents, readout chemistry, and counterstains. Reagentdispensers 26 are preferably bar code labeled 29 for identification bythe computer. For each slide, a single reagent is applied and thenincubated for a precise period of time in a temperature-controlledenvironment. Mixing of the reagents is accomplished by compressed airjets 42 aimed along the edge of the slide thus causing rotation of thereagent. After the appropriate incubation, the reagent is washed off theslide using nozzles 36. Then the remaining wash buffer volume isadjusted using the volume adjust nozzle 39. Coverslip™ solution, toinhibit evaporation, is then applied to the slide via nozzle 37. Airknife 38 divides the pool of Coverslip™ followed by the application ofthe next reagent. These steps are repeated as the carousels turn untilthe protocol is completed.

In addition to host computer 14, apparatus 10 preferably includes itsown microprocessor 44 to which information from host computer 14 isdownloaded. In particular, the computer downloads to microprocessor 44both the sequence of steps in a run program and the sensor monitoringand control logic called the “run rules” as well as the temperatureparameters of the protocol. Model No. DS2251T 128K from DallasSemiconductor, Dallas Tex. is an example of a microprocessor that canperform this function.

Turning now to FIG. 5 there is shown a block diagram of the slideheating system 48. The system generally comprises about twenty thermalplatforms 50, radially mounted to carousel 34, for heating the slidesand monitoring the temperature thereof, and control electronics printedcircuit board 52 also mounted to the slide carousel for monitoring thesensors and controlling the heaters. Control electronics 52 are mountedunder the rotating slide carousel 34. Information and power aretransferred from the fixed instrument platform to the rotating carouselvia a slip ring assembly 56. This information includes the temperatureparameters needed for heating the slides (upper and lower) communicatedfrom microprocessor 44 (after having been downloaded from computer 14)to control electronics 52 as described below. If, during a run, theslide temperature is determined to be below the programmed lower limit,the thermal platform heats the slide. Likewise, if the slide temperatureis found to be above the upper limit, heating is stopped. A power supplyof sufficient capacity to provide about eight watts per heater isprovided to meet the requisite rate of temperature rise (a/k/a “rampup”).

Similarly, in an alternate embodiment, the cooling of the slides may belikewise controlled, as described subsequently. In one alternateembodiment, cooling platforms are mounted below the slide. The coolingplatforms may comprise Peltier-type thermal transducers. In analternative embodiment, a cooling device such as a fan (not shown) mayoptionally be provided if rapid cooling of the slides is required forparticular applications. The cooling device will modify the ambient airfor all of the platforms, necessitating the heaters corresponding to theslides which should not be cooled to compensate for the drop in ambientair temperature. The slide heating system described herein usesconduction heating and heats slides individually. The system providesmore accurate on-slide temperature and allows for temperature settingson a slide by slide basis.

Heating of the Slide

Typically, the biological sample is placed on a top surface of a slide(such as a glass slide). The slide is then placed on top of the thermalplatform 50, so that the bottom surface of the slide is in contact withthe thermal platform. As discussed previously, the thermal platform 50,via conduction, heats the bottom portion of the slide. After the heatingof the biological sample, the inert material may be removed from theslide by a fluid (as a gas or liquid). For example, the inert materialmay be rinsed with de-ionized water and a surfactant. Surfactants usefulin the invention include, but are not limited to, Chemphos TC-227S(Chemron Corp.), Colawet DOSS70 (Colonial Chemical, Inc., SouthPittsburg, Tenn.), Colawet DOSS70E (Colonial Chemical, Inc., SouthPittsburg, Tenn.), Colawet DOSS70PG (Colonial Chemical, Inc., SouthPittsburg, Tenn.), ESI-TERGE™ 320 (CCP/Atofina, Kansas City, Mo.),Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG(Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like.Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG. Morepreferably, the surfactant is sodium dodecylsulfate.

The current method for deparaffinization is markedly different from whatwas performed in the prior art. Prior art methods include: (1) usingorganic solvents to dissolve the paraffin; or (2) manually using heatand dissolving agents to dissolve the paraffin. In contrast, in oneaspect of the current invention, the deparaffinization does not involvea chemical reaction that dissolves the paraffin. In particular, thefluid which is placed on the sample does not solvate the paraffin.Instead, the current method involve the melting of the paraffin from thetissue and the washing of it away with fluids. In one embodiment, thefluid which is placed on top of the embedded sample is not miscible orcapable of being mixed with the paraffin. One example of this is water,which is not miscible with paraffin. In addition, water has a higherdensity than liquified paraffin. The paraffin, when melted, floats tothe top of the fluid so that the top of the slide may be rinsed, rinsingoff of the melted paraffin.

In another method of the present invention, a paraffin embeddedbiological sample is placed on a glass microscope slide and themicroscope slide is placed on a heating element. A reagent is placed onthe biological sample slide, the biological sample slide is then exposedto elevated temperatures that will permit the melting of the inertmaterial, and after which the inert material may be removed from theslide by a fluid (as a gas or liquid).

In a preferred embodiment of the present invention, reagents are used inconjunction with heating the embedded biological samples. Suitablereagents may include, but are not limited to, de-ionized water, citratebuffer (pH 6.0-8.0), Tris-HCl buffer (pH 6-10), phosphate buffer (pH6.0-8.0), SSC buffer, APK Wash™, acidic buffers or solutions (pH 1-6.9),basic buffers or solutions (pH 7.1-14), mineral oil, Norpar, canola oil,and PAG oil. Each of these reagents may also contain ionic or non-ionicsurfactants such as Triton X-100, Tween, Brij, saponin and sodiumdodecylsulfate. Other surfactants useful in the invention include, butare not limited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70(Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70E(Colonial Chemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70PG(Colonial Chemical, Inc., South Pittsburg, Tenn.), ESI-TERGE™ 320(CCP/Atofina, Kansas City, Mo.), Geropon T-77 (Rhodia, Inc.), GeroponAS-200 (Rhodia, Inc.), Hostapon KCG (Clarient Corp.), Monafax 057(Uniquema/ICI Surfactants), and the like. Preferably, the surfactant isChemphos TC-227S or Hostapon KCG. More preferably, the surfactant issodium dodecylsulfate.

In a method of the present invention, the temperature of the heatingelement is raised to a temperature in excess of the melting point of theinert material. For example, the melting point of pure paraffin islisted as 50-57° C. in the Merck index. Thus, in the method of thepresent invention, the temperature is in excess of the melting point ofthe paraffin in which the biological sample is embedded. In a preferredmethod of the present invention, the temperature is raised in excess of50° C. to about 130° C.

In a method of the present invention, the duration of time required tomelt the inert material will vary according to the temperature used andthe embedding material. Typically, in an automated system, a processor,such as a microprocessor, is used in conjunction with a memory. Theamount of time and the temperature required to melt the paraffin iscontained within a table contained in the memory.

The paraffin embedded biological sample is subjected to elevatedtemperatures ranging from 5 minutes to 60 minutes. The heating elementused in the method of the present invention requires that sufficientcontact be maintained between the surface on which the biological sampleis placed and the heating element.

Referring to FIG. 6, there is shown a flow chart for one embodiment forremoving the embedding media from a slide. As shown at block 60, thesurface of the slide is agitated. In one embodiment, the vortex airmixers 42 are used to stir the biological sample on the surface of theslide. As shown at block 62, the temperature for the slide is raised toa predetermined temperature. In one embodiment, the temperature of theslides is raised by heating using the thermal platforms 50. As oneexample for in situ hybridization (ISH), the initial heating step heatsthe slide to 65° C. for 16 minutes and then to 75° C. for 2 minutes. Inan immunohistochemical (IHC) example, initial heating step raises thetemperature of the thermal platforms to 75° C. for approximately 4minutes. This initial heating step, while not necessary to remove theembedding medium, is performed in order to (1) remove any moisture whichis between the biological sample and the surface of the slide; and (2)begin melting the embedding media (the melting point for paraffin is50-57° C., as discussed previously).

The embedding media is both on the outside of the sample (i.e., encasingthe sample) and also infused in the tissue. When performing this initialheating, the embedding media may pool away from the tissue.Specifically, because the slide is at a slide angle (not completelyhorizontal), the embedding media may slowly pool at a point on the slidewhich is lower than where the sample sits. In an alternate embodiment,the slide may be rinsed with a fluid (either liquid or gas) after theinitial heating step to remove the embedding media that has pooled. Forexample, fluids such as de-ionized water may be used to rinse theembedding media from the slide.

As shown at block 64, an aqueous fluid is applied to the slide. Anyaqueous fluid is acceptable such that the fluid covers the entirebiological sample. As discussed previously, examples of aqueous fluidinclude de-ionized water, citrate buffer (pH 6.0-8.0), Tris-HCl buffer(pH 6-10), phosphate buffer (pH 6.0-8.0), SSC buffer, APK Wash™, acidicbuffers or solutions (pH 1-6.9), basic buffers or solutions (pH 7.1-14),mineral oil, Norpar, canola oil, and PAG oil. Moreover, the aqueousfluid may also contain ionic or non-ionic surfactants such as TritonX-100, Tween, Brij, saponin and sodium dodecylsulfate. The surfactantslower the surface tension of the aqueous fluid, allowing the aqueousfluid to spread better over the surface of the slide. In one embodiment,the aqueous fluid comprises de-ionized water with about 0.1% TritonX-100. Other surfactants useful in the invention include, but are notlimited to, Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (ColonialChemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70E (ColonialChemical, Inc., South Pittsburg, Tenn.), Colawet DOSS70PG (ColonialChemical, Inc., South Pittsburg, Tenn.), ESI-TERGE™ 320 (CCP/Atofina,Kansas City, Mo.), Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia,Inc.), Hostapon KCG (Clarient Corp.), Monafax 057 (Uniquema/ICISurfactants), and the like. Preferably, the surfactant is ChemphosTC-227S or Hostapon KCG. More preferably, the surfactant is sodiumdodecylsulfate.

An additional ingredient may be added, acting as an anti-microbialagent, so that the fluid prior to application on the slide does notcontain microbes. In one embodiment, the fluid includes a water content,by weight, of 99% or greater (ie., the fluid is composed of between99%-100% water). The use of water as a fluid to remove the embeddingmaterial is unlike what is conventionally used to remove the embeddingmaterial, such as organic solvents. Water is totally immiscible withparaffin, a typical embedding material. In contrast, organic solvents,such as toluene, xylene, limonene, are miscible with paraffin andtherefore considered suitable for deparaffinization.

Further, the aqueous fluid should be applied in sufficient amounts andat sufficient times (accounting for evaporation of the aqueous fluid dueto heating) such that the embedding media may float to the surface ofthe aqueous fluid and such that the biological sample on the slide willnot dry out. In one embodiment, the aqueous fluid is appliedsequentially, with a first application of approximately 1 mL of aqueousfluid on the biological sample, and with a second application twominutes later of aqueous fluid. The second application may beapproximately 0.5 mL to 1 mL of aqueous fluid. The fluid may be appliedto the slide by using a nozzle which is position directly above theslide. In this manner, the amount of fluid dropped onto the slide may becontrolled. Moreover, because the fluid embedding material (such asparaffin) may have a low surface tension, applying a stream of fluidonto the slide may not leave a sufficient amount of fluid on the top ofthe slide. Thus, using the nozzle to drop the fluid onto the embeddedsample is preferred as it allows more of the fluid to remain on theupper surface of the slide. An additional consideration is themaintenance of the temperature of the biological sample above themelting point of the embedding media. In the paraffin example, themelting point is approximately 50-57° C. In a preferred embodiment, thesample is heated to 75° C. However, since the aqueous fluid is notheated, the application of the aqueous fluid to the slide lowers thetemperature of the biological sample temporarily to approximately 60-65°C., which is still above the melting point of paraffin. Thus, the choiceof temperature to heat the sample should be high enough so that theaddition of fluids to the slide does not lower the temperature of theslide below the melting point of the embedding media. Alternatively, thefluid applied to the slide may be heated prior to application so as notto reduce the temperature of the sample on the slide.

At block 66, an evaporation inhibitor is applied. In a preferredembodiment, LIQUID COVERSLIP™ (LCS™) is applied. Thereafter, the systemwaits for a predetermined amount of time as the slide is heated, asshown at block 68. In in situ hybridization (ISH), one example of thetime for heating the slide is 6 minutes at 75° C. In animmunohistochemical (IHC) example, the incubation period is 8 minutes at75° C. In one embodiment, the temperature of the slides remains constantduring the initial heating until step 76, as discussed subsequently.Thus, the heaters for the slides are turned on during the initial stepof heating and remain on at the same temperature until after theembedding media is rinsed from the slide. Alternatively, the setpointtemperature of the heaters may be adjusted from the time of initialheating until after the embedding media is rinsed from the slide.

During the heating, the embedding media floats to the top of the aqueousfluid. In a preferred embodiment, the fluid which is applied to theslide has density which is greater than the embedding media. In thespecific example, the fluid is mostly composed of water (e.g., 99% orgreater) and the embedding media is paraffin. Paraffin, as an oil basedproduct, is less dense than water. Thus, the paraffin rises to the topof the water after being melted.

The slide is then rinsed, as shown at block 70, carrying away theembedding media in the aqueous fluid. In a preferred embodiment, thesame type of aqueous fluid used to cover the slide is also used to rinsethe slide. The rinse also leaves an amount of aqueous fluid on thebiological sample. In a preferred embodiment, approximately 0.25 mL isleft on the slide. Thereafter, aqueous fluid is further applied to theslide, as shown at block 72. In an alternate embodiment, the rinsing ofthe slide may also leave a sufficient amount of aqueous fluid on theslide such that additional aqueous fluid need not be applied.

At block 74, evaporation inhibitor is applied, which in a preferredembodiment is Liquid Coverslip™ (LCS™). Thereafter, the temperature ofthe slide is reduced, as shown at block 76. In a preferred embodiment,this is done by reducing or eliminating the heat applied by the thermalplatform 50. Moreover, in a preferred embodiment, the temperature of theslide is reduced to a predetermined temperature of 42° C. Thispredetermined temperature is chosen such that all slides, whenprocessing, are at a known temperature (as opposed to being at ambienttemperature, which may fluctuate). Thereafter, the apparatus waits apredetermined amount of time, as shown at block 78.

Another embodiment of the present invention relates to the exposing ofbiological samples without removal of the inert materials in whichbiological samples have been embedded for preservation and support. In apreferred embodiment of the present invention, biological samples arereadied for testing by contact heating of the microscope slide on whichthe embedded biological samples have been placed. Other inert materialsthat are not removed from embedded biological samples include but arenot limited to plastic or celloidin embedding media and/or otherpolymers and resins.

In a preferred method of the present invention, the embedded biologicalsample laying on the glass slide is first heated by the heating element.The heating element exposes heat on one side of the biological sample,such as by using the thermal platforms 50 disclosed in U.S. patentapplication Ser. No. 09/259,240 within an automated staining instrument(U.S. patent application Ser. Nos. 08/995,052 and 60/076,198) such thatthe sample slide is dried.

In another method of the present invention, an embedded biologicalsample is placed on a glass microscope slide and the microscope slide isheated on one side (e.g., by placing the slide on a thermal platform). Areagent is then placed on the biological sample slide and the biologicalsample slide, with the reagent, is then heated to a specifiedtemperature (ranging from ambient to greater than 100° C.) and for aspecified amount of time (ranging from 2 minutes to 12 hours). This willcause etching of the surface of the inert embedding material, and afterwhich the etching reagent may be removed from the slide by a fluid (as agas or liquid). As discussed previously, the amount of time and thespecified temperature may be stored in memory.

In the preferred method of the present invention, reagents are used inconjunction with or without heating the embedded biological samples.Suitable reagents may include, but are not limited to, de-ionized water,citrate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 6-10), phosphate buffer(pH 6.0-8.0), SSC buffer, APK Wash™, acidic buffers or solutions (pH1-6.9), basic buffers or solutions (pH7.1-14) mineral oil, Norpar,canola oil, and PAG oil. Each of these reagents may also contain ionicor non-ionic surfactants, which include, but are not limited to,Chemphos TC-227S (Chemron Corp.), Colawet DOSS70 (Colonial Chemical,Inc., South Pittsburg, Tenn.), Colawet DOSS70E (Colonial Chemical, Inc.,South Pittsburg, Tenn.), Colawet DOSS70PG (Colonial Chemical, Inc.,South Pittsburg, Tenn.), ESI-TERGE™ 320 (CCP/Atofina, Kansas City, Mo.),Geropon T-77 (Rhodia, Inc.), Geropon AS-200 (Rhodia, Inc.), Hostapon KCG(Clarient Corp.), Monafax 057 (Uniquema/ICI Surfactants), and the like.Preferably, the surfactant is Chemphos TC-227S or Hostapon KCG. Morepreferably, the surfactant is sodium dodecylsulfate.

In the method of the present invention, the temperature of the heatingelement is set to an appropriate level for the drying or the etching ofthe embedded biological sample. For example, etching may be carried outwith a basic solution of methanol sodium hydroxide (sodium methoxide) attemperatures ranging from ambient to 37° C.

In the method of the present invention, the duration of time required toetch the inert material will vary according to the temperature used andthe embedding material (plastic or celloidin embedding media and/orother polymers and resins, etc.). In a preferred method of the presentinvention the embedded biological sample is subjected to appropriatetemperatures ranging from 2 minutes to 12 hours. The heating elementused in the method of the present invention requires that sufficientcontact be maintained between the surface on which the biological sampleis placed and the heating element.

A preferred embodiment of the present invention also comprises anautomated method of cell conditioning, either concurrent with,subsequent to or independent of removal or etching of the inertembedding material from the biological sample. Heating the biologicalsample in an appropriate (organic or inorganic) reagent has been foundto improve the accessibility of the reagent to the target molecule inthe cell (protein, nucleic acid, carbohydrate, lipid, pigment or othersmall molecule, etc.). This process of improving accessibility of thereagent (organic or inorganic) to the molecular target is referred toherein as cell conditioning.

In one method of the present invention, cell conditioning isaccomplished while the biological sample is being exposed as describedabove. In this method of the present invention, a biological sample isplaced on a glass microscope slide and the microscope slide is heated onone side (e.g., by placing the slide on a thermal platform) within anautomated staining instrument (U.S. patent application Ser. Nos.08/995,052 and 60/076,198). A reagent is placed on the biological sampleand the temperature of the heating element may or may not be increased.The biological sample is exposed to the appropriate temperature for anappropriate duration of time that will permit the melting or etching ofthe inert material and permit cell conditioning of the biological sampleto be subsequently stained using histological or cytological techniques.

Referring to FIGS. 7 a-b, there are shown flow charts of one embodimentfor exposing a biological sample to permit cell conditioning. As shownat block 80, it is determined whether cell conditioning is applied. Avariety of cell conditioners may be applied depending on the processingnecessary. As shown at block 82, the slide is rinsed with aqueous fluid.Any aqueous fluid is acceptable such that the fluid covers the entirebiological sample. In a preferred embodiment, de-ionized water withTriton X-100™ is used. Thereafter, an evaporation inhibitor is appliedto the slide, as shown at block 84. In a preferred embodiment, LiquidCoverslip™ is applied. Thereafter, the temperature of the slide ismodified to a predetermined value, as shown at block 86. In a preferredembodiment, heaters heat the slide to 42° C. This initial heating isperformed in order to ensure that all of the slides begin the cellconditioning at a predetermined temperature; otherwise, the temperatureof the slide may be unknown if the temperature of the slides isdetermined by the ambient temperature.

Evaporation inhibitor is then applied, as shown at block 88. Thereafter,the temperature of the slides is raised to a predetermined temperature,as shown at block 90. This is the temperature at which the cellconditioning is performed. In a preferred embodiment, the temperature ofthe heaters is set to 100° C. Thereafter, cell conditioner is applied,as shown at block 92 and evaporation inhibitor is applied, as shown atblock 94. A multitude of cell conditioners may be applied, as discussedherein.

How lightly/heavily a sample is fixed determines the amount of cellconditioning necessary. If a sample is lightly fixed, a mild cellconditioning is recommended. Likewise, if the sample is moderately orheavily (extended) fixed, a moderate or a heavy cell conditioning,respectively, is recommended. Improper cell conditioning may haveadverse consequences on the processing of the sample. In a preferredembodiment, cell conditioning mild/medium/heavy time is 30/60/90 minutesrespectively. Moreover, the cell conditioner is applied at predeterminedincrements within the processing in order to properly condition the celland to avoid drying out the sample. As shown in FIG. 7 a, the number oftimes to iterate through the application of cell conditioner and LCS™ isdetermined, as shown at block 96. This variable is called“loop_counter.” Loop_counter is set to 0, as shown at block 98 in FIG. 7b. A loop is entered (as shown at block 100) and the system waits apredetermined amount of time (as shown at block 102). In a preferredembodiment, the predetermined amount of time is 6 minutes. Cellconditioner is applied (as shown at block 104) and evaporation inhibitoris applied (as shown at block 106). The loop_counter is thenincremented, as shown at block 108. In a preferred embodiment, thenumber of times the loop is executed is 5/10/15 for mild/medium/heavycell conditioning. Thereafter, the temperature of the slide is reduced,as shown at block 110. In a preferred embodiment, the heater temperatureis reduced to 42° C. Alternatively, the heater may be turned off and theslide temperature may revert to the ambient temperature. Thereafter, theslide is rinsed, as shown at block 112.

The reagents used for cell conditioning can be the same as those forexposing the embedded biological sample. For example, for DNA targets, acell conditioning solution may be a solution of EDTA; a commontemperature setting may be 95° C. for a duration ranging from 2-90minutes. For protein targets, a cell conditioning solution may be asolution of boric acid buffer; a common temperature setting may be inexcess of 100° C. for a duration ranging from 2-90 minutes. For RNAtargets, a cell conditioning solution may be a solution of SSC; a commontemperature setting may be 75° C. for a duration ranging from 2-90minutes. For histochemical reactions, such as a Hematoxylin and Eosin(H&E) stain, a cell conditioning solution may be treated de-ionizedwater; a common temperature may range from 60-S0° C. for a duration of2-90 minutes. A partial list of possible reagents appears in AnalyticalMorphology, Gu, ed., Eaton Publishing Co. (1997) at pp. 1-40. Thesolutions should generally be of known molarity, pH, and composition.Sodium dodecyl sulfate (SDS) and/or ethylene glycol is preferably addedto the conditioning solution. In addition, metal ions or other materialsmay be added to these reagents to increase effectiveness of the cellconditioning.

In another method of the present invention, cell conditioning isaccomplished subsequent to the biological sample being exposed asdescribed above. In this method of the present invention a biologicalsample is placed on a glass microscope slide and the microscope slide isheated on one side (e.g., by placing the slide on a thermal platform)within an automated staining instrument (U.S. patent application Ser.Nos. 08/995,052 and 60/076,198). In this method (one embodiment of whichis shown in FIG. 6), the embedded biological sample laying on the glassslide is first heated by the heating element within an automatedstaining instrument such that the sample slide is dried and theembedding material is melted or etched and removed by the application ofa fluid. Subsequent to exposing the biological sample, an appropriatereagent is applied in order to permit cell conditioning of thebiological sample to be subsequently stained using histological orcytological techniques.

The reagents used for cell conditioning can be the same as those forexposing the embedded biological sample. For example, for DNA targets, acell conditioning solution may be a solution of SSC; a commontemperature setting may be 95° C. for a duration ranging from 2-90minutes. For protein targets, a cell conditioning solution may be asolution of phosphate buffer; a common temperature setting may be inexcess of 100° C. for a duration ranging from 2-90 minutes. For RNAtargets, a cell conditioning solution may be a solution of SSC; a commontemperature setting may be 75° C. for a duration ranging from 2-90minutes. For histochemical reactions, such as a Trichrome stain, a cellconditioning solution may be Bouins; a common temperature may range from60-80° C. for a duration of 2-30 minutes.

In yet another method of the present invention, cell conditioning isaccomplished without the biological sample being exposed. In this methodof the present invention, a biological sample is placed on a glassmicroscope slide and the microscope slide placed on a heating elementwithin an automated staining instrument. A reagent is placed on thebiological sample and the temperature of the heating element may or maynot be increased. Cell conditioning of the biological sample may beperformed prior to being stained using histological or cytologicaltechniques.

The reagents used for cell conditioning can be the same as those forexposing the embedded biological sample. For example, for DNA targets, acell conditioning solution may be a solution of sodium citrate; a commontemperature setting may be 90° C. for a duration ranging from 2-90minutes. For protein targets, a cell conditioning solution may be asolution of urea; a common temperature setting may be in excess of 100°C. for a duration ranging from 2-90 minutes. For whole cells, a cellconditioning solution may be a solution of methanol; a commontemperature setting may be ambient for a duration ranging from 4-10minutes. For histochemical reactions, such as an Acid Fast Bacilli (AFB)stain, a cell conditioning solution may be peanut oil; a commontemperature may range from 60-70° C. for a duration of 30-60 minutes.

The present invention also comprises cell conditioning of cytologicalpreps, such as fine needle aspirations (FNA) smears, touch preps,Ficoll, Cytospins®, Thins Preps®, cervical-vaginal pap smears, blood orbody fluid films, etc., that are neither fixed with an aldehyde norembedded in a matrix, such as paraffin. Many are fixed in an alcohol,such as methanol or ethanol, others will be sprayed with hair spray orother aerosol fixative and dried, and still others will be placed incytological fixatives, which may include carbowax and Saccomanno's(organic or inorganic) reagent among others. The cells are eithercentrifuged or filtered to a slide or directly touched to a glass slideand smeared in some cases (PAP's) or applied directly against the slide(touch preps).

The term “Biological sample” includes, but is not limited to, anycollection of cells (either loose or in tissue) that can be mounted on astandard glass microscope slide including, without limitation, sectionsof organs, tumors sections, bodily fluids, smears, frozen sections,blood, cytology preps, microorganisms, tissue arrays and cell lines.

The term “Stain” includes, but is not limited to, any biological orchemical entity which, when applied to targeted molecules in biologicalsample, renders the molecules detectable under microscopic examination.Stains include without limitation detectable nucleic acid probes,antibodies, and other reagents which in combination or by themselvesresult in a colored end product (by bright field or fluorescence).

The terms “Reagent”, “Buffer”, “Additive”, “Component” and “Solution” asused herein for exposing or deparaffinizing (i.e., the process ofdeparaffinization) may comprise the following component or components,all of which are available from Sigma Chemical, unless otherwise noted:de-ionized water, de-ionized water with about 0.1% Triton X-100, 10 mMphosphate at around pH 6.1, 10 mM phosphate with about 0.1% Triton X-100at around pH 6.1, 10 mM citrate at around pH 6, 10 mM citrate with about0.1% Triton X-100, 2×SSC, 10 mM Tris[hydroxymethyl]aminomethane chloride(i.e., Tris-Cl) at around pH 8.2, 10 mM Tris-CJ with about 0.1% TritonX-100 at around pH 8.2. A person skilled in the art to which thisinvention pertains will recognize that the concentration orconcentrations of the component or components listed above may be variedwithout altering the characteristics of the reagent, buffer, additive orsolution for exposing or deparaffinizing.

The terms “Reagent”, “Buffer”, “Additive”, “Component”, “Solution” and“Cell Conditioner” as used herein for cell conditioning may comprise thefollowing component or components, all of which are available from SigmaChemical, unless otherwise noted: 5 mM citrate at around pH 6, 5 mMcitrate with about 0.5% sodium dodecyl sulfate (SDS) at around pH 6, 10mM citrate at around pH 6, 10 mM citrate with about 0.5% SDS at aroundpH 6, 20 mM citrate at around pH 6, 20 mM citrate with about 0.5% SDS ataround pH 6, 50 mM citrate at around pH 6, 50 mM citrate with about 0.5%SDS at around pH 6, 1 mM ethylene diamine tetraacetic acid (EDTA) ataround pH 8, 1 mM EDTA with about 0.075% SDS at around pH 8, 10 mM EDTAat around pH 8, 10 mM EDTA with about 0.075% SDS at around pH 8, 20 mMEDTA at around pH 8, 20 mM EDTA with about 0.075% SDS at around pH 8, 50mM EDTA at around pH 8, 50 mM EDTA with about 0.075% SDS at around pH 8,10 mM citrate with about 0.5% SDS and about 1% ethylene glycol at aroundpH 6, 10 mM citrate with about 0.5% SDS and about 5% ethylene glycol ataround pH 6, 10 mM citrate with about 0.5% SDS and about 10% ethyleneglycol at around pH 6, 1 mM EDTA with about 0.075% SDS and about 1%ethylene glycol at around pH 8, 1 mM EDTA with about 0.075% SDS andabout 5% ethylene glycol at around pH 8, 1 mM EDTA with about 0.075% SDSand about 10% ethylene glycol at around pH 8, phosphate/citrate/EDTA atabout pH 9, 10 mM citrate with about 10 mM urea at around pH 6, 10 mMcitrate with about 1 mM urea at around pH 6.2, 10 mM sodium citrate withabout 1.4 mM MgCl₂ and about 0.1% SDS at around pH 7, 10 mM sodiumcitrate with about 1.4 mM MgCl₂ and about 0.1% SDS at around pH 7.99, 10mM Tris-Cl at around pH 8, 10 mM Tris-Cl with about 20% formamide ataround pH 8, 10 mM citrate with about 5% dimethyl sulfoxide (DMSO) ataround pH 6, 10 mM citrate with about 0.1% Triton X-100 and about 20%formamide at around pH 6, 10 mM phosphate with 5×SSC and about 2.5%chrondroitin A at around pH 7, 10 mM Tris-Cl with about 10 mM EDTA andabout 0.1% Triton X-100 and about 20% formamide at around pH 8.2, 10 mMcitrate with about 20% glycerol at around pH 6, 10 mM citrate with about0.1% Triton X-100 and about 10 mM glycine at around pH 6, 1 mM EDTA withabout 1 mM citrate and about 0.25% SDS at around pH 7.8, Norpar/mineraloil (high temperature coverslip), PAG-100 oil, 10 mM citrate with about2% SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 1%SDS at a pH of around 6 to around 6.2, 10 mM citrate with about 0.5% SDSat a pH of around 6 to around 6.2, 10 mM citrate with about 0.25% SDS ata pH of around 6 to around 6.2, 1 mM EDTA with about 2% SDS at a pH ofaround 7.5 to around 8, 1 mM EDTA with about 1% SDS at a pH of around7.5 to around 8, 1 mM EDTA with about 0.5% SDS at a pH of around 7.5 toaround 8, 1 mM EDTA with about 0.25% SDS at a pH of around 7.5 to around8, 1 mM EDTA with about 0.1% SDS at a pH of around 7.5 to around 8, 1 mMEDTA with about 0.075% SDS at a pH of around 7.5 to around 8, 0.5 mMEDTA with about 0.25% SDS at around pH 8, 10 mM EDTA with about 0.5% SDSat around pH 9.6. A person skilled in the art to which this inventionpertains will recognize that the concentration or concentrations of thecomponent or components listed above may be varied without altering thecharacteristics of the reagent, buffer, additive or solution for cellconditioning.

A preferred embodiment of the present invention is the basic formulationcomprising 10 mM Tris base, 7.5 mM boric acid, 1 mM EDTA (disodiumsalt), 0.05% ProClin™ 300 (Supelco, Inc., Bellefonte, Pa.) at pH 8.5.Other embodiments contemplated include the basic formulation wherein theTris base concentration ranges from about 5 mM to about 20 mM, whereinthe boric acid concentration ranges from about 5 mM to about 40 mM,wherein the EDTA concentration ranges from about 0.5 mM to about 2 mM orwherein the pH ranges from around pH 7 to around pH 9. Temperatureranges contemplated are from around 95° C. to around 100° C. Withoutintending to be construed as a limitation, it should be noted that nosignificant difference is seen when the concentration of Tris base orEDTA varies over the range specified above, significantly better resultswere obtained when the concentration of boric acid was 10 mM as opposedto 20 mM or 40 mM, slightly better results were obtained when theconcentration of boric acid was 10 mM as opposed to 5 mM, conditioningis optimal at a pH of around 8 to around 8.6. Comparing 10 mM Tris+20 mMboric acid+1 mM EDTA+0.5% Brij 35 with 10 mM Tris+20 mM boric acid+0.5%Brij 35, both reagents displayed strongest conditioning at 100° C. withsignificantly weaker conditioning at 95° C.

Other embodiments contemplated include, but are not limited to, citratebuffer (a combination of sodium citrate trisodium salt dihydrate andcitric acid monohydrate hydrate), 10 mM Tris+20 mM boric acid+1 mM EDTA,10 mM Tris+20 mM boric acid, 10 mM Tris+1 mM EDTA, 10 mM Tris, 20 mMboric acid, 1 mM EDTA, 20 mM Tris+20 mM boric acid+1 mM EDTA, 5 mMTris+20 mM boric acid+1 mM EDTA, 10 mM Tris+20 mM boric acid+2 mM EDTA,10 mM Tris+20 mM boric acid+0.5 mM EDTA, 10 mM Tris+40 mM boric acid+1mM EDTA, 10 mM Tris+10 mM boric acid+1 mM EDTA, 10 mM Tris+5 mM boricacid+1 mM EDTA, 10 mM Tris+7.5 mM boric acid+1 mM EDTA, 10 mM Tris+20 mMboric acid+1 mM EDTA+5% ethylene glycol, 10 mM Tris+20 mM boric acid+5%ethylene glycol, 10 mM Tris+20 mM boric acid+1 mM EDTA+0.1% SDS, 10 mMTris+20 mM boric acid+0.1% SDS, 10 mM Tris+20 mM boric acid+1 mM EDTA+5%DMSO, 10 mM Tris+20 mM boric acid+5% DMSO, 10 mM Tris+20 mM boric acid+1mM EDTA+10% DMSO, 10 mM Tris+20 mM boric acid+10% DMSO, 10 mM Tris+20 mMboric acid+1 mM EDTA+5% formamide, 10 mM Tris+20 mM boric acid+5%formamide, 10 mM Tris+20 mM boric acid+1 mM EDTA+10% formamide, 10 mMTris+20 mM boric acid+10% formamide, 10 mM Tris+20 mM boric acid+1 mMEDTA+0.5% Brij 35, 10 mM Tris+20 mM boric acid+0.5% Brij 35, 10 mMTris+20 mM boric acid+1 mM EDTA+0.1% Brij 35, 10 mM Tris+20 mM boricacid+0.1% Brij 35, 10 mM Tris+20 mM boric acid+1 mM EDTA+0.5% TritonX-100, 10 mM Tris+20 mM boric acid+0.5% Triton X-100.

The term “10×SSC” refers to a 10 molar concentration of sodiumchrloide/sodium citrate solution, comprising deionized water as neededto make a liter solution, 87.66 g NaCl, 44.12 g citric acid trisodiumsalt, dihydrate, adjusted to pH 7.0 with HCl or NaOH, as appropriate.0.5 ml ProClin 300 is added as preservative. For all phosphate buffersprepared at any concentration (X molar), prepare X molar solutions of(1) HPO₄ ⁻² using K₂HPO₄ or NaHPO₄, and (2) H₂PO₄ ⁻ using KH₂PO₄ orNaH₂PO₄.

Another preferred embodiment of the cell conditioner (cell conditioningsolution 2 or CC2) comprises about 10 mM citrate buffer at about pH 6,about 5% ethylene glycol, about 1 mM sodium metabisulfite (Morphosave™,Ventana Medical Systems, Inc., Tucson, Ariz.; U.S. Pat. No. 5,432,056)and about 0.3% sodium dodecyl sulfate (SDS). The concentration ofcitrate may range from about 5 mM to about 50 mM. The pH of the buffermay range from about 4 to about S. The concentration of ethylene glycolmay range from about 1% to about 10%. The concentration of sodiummetabisulfite may range from about 0.1 mM to about 10 mM, preferablyfrom about 0.5 mM to about 1.5 mM. The concentration of SDS may rangefrom about 0.1% to about 1%, preferably from about 0.25% to about 0.5%.

The following examples are presented for illustrative purposes only andare not intended, nor should they be construed, as limiting theinvention in any way. Those skilled in the art will recognize thatvariations on the following can be made without exceeding the spirit orscope of the invention. All patents, patent applications, and otherpublications are hereby incorporated by reference in their entirety.

EXAMPLES Example 1 Automated “Exposing” and “Cell Conditioning” withBiological Samples Stained with H&E

Biological samples, including breast, stomach, brain, tonsil and kidney,that had been embedded in paraffin were exposed according to thefollowing procedure: slides containing the above referenced biologicalsample were placed on an automated instrument (Ventana Medical Systems,Inc., Tucson, Ariz.) and subjected to the exposing protocol describedbelow. Generally, the slides containing paraffin embedded biologicalsamples were dry heated to 65° C. for six (6) minutes then rinsed withany of the following: 1) 1× citrate buffer, 2) de-ionized water, 3) 10mM phosphate buffer (pH=6.3), or 4) 10 mM Tris-HCl buffer (pH=7.4) eachcontaining 0.1% Triton X-100.

Exposing Protocol 1

-   -   1. Incubate for 2 minutes    -   2. Rinse slide    -   3. Adjust slide volume and apply LIQUID COVERSLIPLIQUID        COVERSLIP™    -   4. Incubate for 6 minutes    -   5. Rinse slide    -   6. Adjust slide volume and apply LCS™    -   7. Increase temperature to 65.0° C.    -   8. Rinse slide    -   9. Adjust slide volume and apply LCS™    -   10. Incubate for 4 minutes    -   11. Adjust slide volume and apply LCS™    -   12. Incubate for 4 minutes    -   13. Adjust slide volume and apply LCS™    -   14. Incubate for 4 minutes    -   15. Rinse slide    -   16. Decrease temperature to 42.0° C.    -   17. Adjust slide volume and apply LCS™    -   18. Incubate for 4 minutes    -   19. Rinse slide    -   20. Decrease temperature to 42.0° C.    -   21. Adjust slide volume and apply LCS™    -   22. Incubate for 4 minutes    -   23. Rinse slide

After automated exposing, the biological sample was stained withhematoxylin and eosin by the following method. Slides were placed inhematoxylin 1 (Richard Allen Scientific, Kalamazoo, Mich.) for 1.5minutes and then rinsed with running de-ionized water for one minute.Slides were then placed in acid alcohol clarifier (Richard AllenScientific) for one minute and then rinsed with running de-ionized waterfor one minute. Slides were then placed in diluting ammonia-bluingreagent for one minute (Richard Allen Scientific, Kalamazoo, Mich.) andthen rinsed in running de-ionized water for one minute. Slides were thenrinsed in 95% ethanol, and then placed in 2.5% eosin Y (Richard AllenScientific, Kalamazoo, Mich.) for 2.5 minutes. The biological samples onthe slides were dehydrated by exposing the biological sample to a 100%ethanol bath for one minute. This process was repeated three timesfollowed by exposure of the biological sample to a xylene bath for threeminutes, twice. After the dehydration step the biological sample wascovered with a coverslip.

Control biological samples were deparaffinized by a traditionalsolvent-based deparaffinization technique. Paraffin-embedded biologicalsamples placed on microscope slides and preserved in paraffin werecompletely submersed in a xylene bath for five minutes. Slidescontaining biological samples were placed in a second xylene bath forfive minutes. After removal from the second xylene bath, the slides wereplaced in a 100% ethanol bath for three minutes. Slides were then placedin a second 100% ethanol bath for three minutes and then placed in a 90%ethanol solution for two minutes. The slides were then placed in 80%ethanol for one minute followed by complete immersion in distilled waterfor one to three minutes. After deparaffinization, the biologicalsamples were stained with hematoxylin and eosin as described above.

The biological samples that were deparaffinized by the solvent techniqueand by the automated heating technique were compared after staining byhematoxylin and eosin. Morphology on all sets of slide was acceptableand essentially equivalent The tonsil and brain biological samples thatwere exposed by the automated heating method showed more intensifiedhematoxylin staining than the biological samples deparaffinized bystandard solvent techniques.

Example 2 Automated “Exposing” of Biological Samples with Simultaneous“Cell Conditioning”

Biological samples of kidney and tonsil that had been formalin fixed andembedded in paraffin were exposed according to the protocol described inExample 1. After automated exposing, the biological sample was subjectedto the automated DAB paraffin protocol used for immunohistochemicalstaining. The protocol for DAB staining is described below:

DAB Protocol

-   -   1. Incubate for 2 minutes    -   2. Rinse slide    -   3. Adjust slide volume and apply LCS™    -   4. Rinse slide    -   5. Adjust slide volume and apply LCS™    -   6. Rinse slide    -   7. Adjust slide volume and apply LCS™    -   8. Apply one drop of inhibitor    -   9. Incubate for 4 minutes    -   10. Adjust slide volume and apply LCS™    -   11. Apply one drop of primary antibody    -   12. Incubate for 32 minutes    -   13. Adjust slide volume and apply LCS™    -   14. Apply one drop of Biotinylated Ig    -   15. Incubate for 8 minutes    -   16. Rinse slide    -   17. Adjust slide volume and apply LCS™    -   18. Apply one drop of Avidin-HRPO    -   19. Incubate for 8 minutes    -   20. Rinse slide    -   21. Adjust slide volume and apply LCS™    -   22. Apply one drop of DAB and one drop DAB H₂O₂    -   23. Incubate for 8 minutes    -   24. Rinse slide    -   25. Adjust slide volume and apply LIQUID COVERSLIP™    -   26. Apply one drop of Copper    -   27. Incubate for 4 minutes    -   28. Rinse slide

The primary antibody used for the kidney biological sample was Anti-CD15(Ventana Medical Systems, Inc. Tucson, Ariz., Catalogue no. 250-2504).The primary antibody used for the tonsil biological sample wasAnti-CD45RO (Ventana Medical Systems, Inc. Tucson, Ariz., Catalogue no.250-2563). The DAB staining kit used was obtained from Ventana MedicalSystems, Inc. Tucson, Ariz., Catalogue no. 250-001.

Control biological samples were deparaffinized by a traditionalsolvent-based deparaffinization technique, as described in Example 1.After deparaffinization the biological samples were placed in a pressurecooker (Model #62104 Nordic Ware, Minneapolis, Minn.) containing 1.5 L1× citrate buffer. The pressure cooker was then sealed and placed in amicrowave oven (Model #MQSO836E, Matsushita, Franklin Park, Ill.). Withthe microwave oven set on “high,” the samples were subjected tomicrowave heating for approximately 30 minutes. After microwaving thesamples were then “cured” for 30 minutes in the pressure cooker with thelid securely fastened. After curing the biological samples were placedin 1× citrate buffer for two minutes. The biological samples were thenremoved from the citrate buffer and the end of the slides blotted toremoved excess citrate buffer. After blotting, the slides were placed onthe automated instrument and immunohistochemically stained as describedabove.

The biological samples deparaffinized by the solvent technique and bythe automated exposing and simultaneous cell conditioning technique werecompared after immunohistochemical staining. Morphology and staining onall sets of slides was acceptable and essentially equivalent.

Example 3 Two Step Automated “Exposing” and “Cell Conditioning”

Biological samples of tonsil and breast that had been preserved inparaffin and treated with formaldehyde were treated by the followingprotocol:

Exposing and Cell Conditioning Protocol

-   -   1. Incubate for 2 minutes    -   2. Increase thermofoil temperature to 65.0° C.    -   3. Incubate for 6 minutes    -   4. Rinse slide and apply LCS™    -   5. Incubate for 6 minutes    -   6. Rinse slide and apply LCS™    -   7. Increase thermofoil temperature to 100.0° C.    -   8. Adjust slide volume and apply LCS™    -   9. Rinse slide    -   10. Adjust slide volume and apply LCS™    -   11. Incubate for 4 minutes    -   12. Adjust slide volume and apply LCS™    -   13. Incubate for 4 minutes    -   14. Adjust slide volume and apply LCS™    -   15. Incubate for 4 minutes    -   16. Adjust slide volume and apply LCS™    -   17. Incubate for 4 minutes    -   18. Adjust slide volume and apply LCS™    -   19. Incubate for 4 minutes    -   20. Adjust slide volume and apply LCS™    -   21. Incubate for 4 minutes    -   22. Adjust slide volume and apply LCS™    -   23. Incubate for 4 minutes    -   24. Adjust slide volume and apply LCS™    -   25. Incubate for 4 minutes    -   26. Adjust slide volume and apply LCS™    -   27. Incubate for 4 minutes    -   28. Rinse slide    -   29. Decrease temperature to 42.0° C.    -   30. Adjust slide volume and apply LCS™    -   31. Incubate for 4 minutes    -   32. Rinse slide    -   33. Decrease temperature to 20.0° C.    -   34. Adjust slide volume and apply LCS™    -   35. Incubate for 4 minutes    -   36. Rinse slide

The buffer used in the protocol was SSC buffer with either 20% formamideor 0.1% Triton. After the biological sample was subjected to the aboveprotocol, the DAB paraffin protocol used for immunohistochemicalstaining of Example 2 was applied. Tonsil biological sample was treatedwith anti-Ki67 as a primary antibody. Breast samples were treated withanti-estrogen receptor (6F11) or anti-progesterone receptor (1A6) as aprimary antibody. All primary antibodies are available through Ventana.

Control biological samples were deparaffinized by a traditionalsolvent-based de-paraffinization technique, as described in Example 1.After deparaffinization the biological samples were placed in a pressurecooker (Model #62104 Nordic Ware, Minneapolis, Minn.) containing 1.5 L1× citrate buffer. The pressure cooker was then sealed and placed in amicrowave oven (Model #MQSO836E, Matsushita, Franklin Park, Ill.). Withthe microwave oven set on “high”, the samples were subjected tomicrowave heating for approximately 30 minutes. After microwaving thesamples were then “cured” for 30 minutes in the pressure cooker with thelid securely fastened. After curing the biological samples were placedin 1× citrate buffer for two minutes. The biological samples were thenremoved from the citrate buffer and the end of the slides were blottedto removed excess citrate buffer. After blotting the slide were placedon the automated instrument and immunohistochemically stained asdescribed above.

The biological samples deparaffinized by solvent technique and by theautomated heating technique were compared after immunohistochemicalstaining. Morphology on all sets of slide was acceptable and essentiallyequivalent. Staining with Ki67 on all sets of slides was equivalent. ER(6F11) and PR (1A6) staining was slightly weaker with automated cellconditioning indicating the process does work but more development andoptimization is required.

Example 4 Automated Cell Conditioning of Non Paraffin Embedded CellLines for in situ hybridization Thin Preps™

Hela (ATCC lot # 980427H), Caski (ATCC lot # 980416C) and Siha (ATCC lot# 980416S) cell lines stored in Cytyk preparation solution (lot #01139Q) were deposited on microscope slides using the Cytyk 2000instrument. After deposition the slides were placed in alcohol to keepmoist until use on the Discovery® In-Situ staining module (VentanaMedical Systems Inc., Tucson, Ariz.). Slides were loaded into theinstrument and wetted with 2×SSC made from 20×SSC (Ventana P/N 650-012).Slides were run through a cell conditioning protocol currently referredto as Depar 30 where the slides are rinsed with 2×SSC and thetemperature of the slides is increased to 95° C. for a period ofapproximately 30 minutes. The slides are then cooled to 37° C. andrinsed with APK Wash® prior to the in-situ staining run.

Using the protocol Blue Swap ISH the cell lines were stained for HPV16/18 (Enzo HPV 16/18 Bio Probe cat # 32874). Prior to probe applicationthe cell lines are enzymatically digested with Protease 2 (Ventana P/N250-2019). After the probe application the probe and biological sampleare denatured simultaneously at 95° C. for 8 minutes. Thenon-specifically bound probe is washed off with stringency washes of2×SSC at 55° C. The probe is then detected with Streptavidin Alk Phosand NBT/BCIP.

The cell lines were dehydrated after staining with a one-minute exposureto 95% ethanol and a one-minute exposure to 100% ethanol repeated 2times. Following the ethanol the slides were exposed to xylene for 3minutes twice. After dehydration the slides were coverslipped.

The stained cell lines after conditioning showed acceptable morphology,weak staining and there was high background on these slides indicating aneed for the process to be developed more.

Depar 30 Protocol

Wet Load Slides

-   -   1. Skip Application & Incubate for 2 minutes    -   2. Rinse Slides (2×SSC Buffer) (Warm Slides to 65° C.)    -   3. Adjust Slide Volume, then apply LCS™    -   4. Skip Application & Incubate 6 minutes    -   5. Rinse Slides (2×SSC Buffer) (Warm Slides to 95° C.)    -   6. Adjust Slide Volume, then Apply LCS™    -   7. Rinse Slides    -   8. Adjust Slide Volume, then Apply LCS™    -   9. Skip Application & Incubate for 4 minutes    -   10. Adjust Slide Volume, then Apply LCS™    -   11. Skip Application & Incubate for 4 minutes    -   12. Adjust Slide Volume, then Apply LCS™    -   13. Skip Application & Incubate for 4 minutes    -   14. Adjust Slide Volume, then Apply LCS™    -   15. Skip Application & Incubate for 4 minutes    -   16. Adjust Slide Volume, then Apply LCS™    -   17. Skip Application & Incubate for 4 minutes    -   18. Adjust Slide Volume, then Apply LCS™    -   19. Skip Application & Incubate for 4 minutes    -   20. Adjust Slide Volume, then Apply LCS™    -   21. Skip Application & Incubate for 4 minutes    -   22. Rinse Slides (2×SSC Buffer) (Warm Slides to 37° C.)    -   23. Adjust Slide Volume, then Apply LCS™    -   24. Skip Application & Incubate for 4 minutes    -   25. Rinse Slides (APK Wash)    -   26. Adjust Slide Volume, then Apply LCS™

Example 5 Automated “Exposing” and “Cell Conditioning” for Single CopyDNA Detection

Slides containing formalin fixed, paraffin embedded cell lines Caski(R96-1050A) and Siha (R96-96-C2), both generously provided by Dr.Raymond Tubbs, Cleveland Clinic Pathology Dept., Cleveland Ohio, werestained on Ventana target slides. Slides were dry loaded onto theinstrument and the slide temperature was increased to 65° C. The Depar30 protocol was run wherein the slides are rinsed with 2×SSC Bufferwhile at 65° C. then the heat is increased to 95° C. for about 40minutes. The slides were then cooled to 37° C. and rinsed with APK wash.At this time the following in situ protocol was run:

In situ Protocol: Tubbs 1

-   -   (Ventana APK Wash was used for all rinse steps)    -   Protease Digestion: Protease 2, 4 minutes, 37° C.    -   Inhibitor Step: Ventana Inhibitor from DAB kit 32 minutes 37° C.    -   Probe: Enzo HPV Bio Probe 16/18    -   Control Probe: Enzo HPV Bio Probe 6/11    -   Denaturation: 95° C., 8 minutes    -   Hybridization: 37° C., 64 minutes    -   2 Stringency Washes 2×SSC, 60° C., 8 minutes each    -   3^(rd) Stringency Wash 2×SSC, 37° C., 4 minutes    -   Probe Detection: Streptavidin HRPO (Dako GenPoint Cat. #K0620)    -   Amplification: Biotinyl Tyramide (Dako GenPoint Cat. # K0620)    -   Detection: Streptavidin HRPO (Dako GenPoint Cat. #K0620) or    -    Streptavidin Alk Phos (Vector Cat. # SA5100)    -   Chromogen DAB (Dako Gen Point Cat. # K0620) or    -    Ventana NBT/BCIP (Kit P/N250-060) or    -    Ventana Naphthol/Fast Red (Kit P/N250-030)

Example 6 Automated “Cell Conditioning” for Non-paraffin EmbeddedSamples

The protocol for DAB staining as described in Example 2 was used in thisExample.

The cell conditioning steps for these antibodies was done after using aCytyk 2000® instrument to make ThinPreps® of cell lines. The ThinPreps®were stained using antibodies to ER, PgR, Ki67, P53 on Ventana ESinstruments, NexES instruments and a manual procedure (Cytyk, Inc.). Aduplicate group of slides have been stained on the NexES Insita module,allowing the cell conditioning steps to be performed by automation.

Although the example stated above is specific to the Cytyk® instrumentand staining of the ThinPreps®, the experience is not limited to thatmode of making cytological preps.

Example 7 “Cell conditioning” of Frozen Biological Sample

Frozen tonsil blocks were prepared by cutting six sections from eachblock and placing the sample on microscope slides. Four slides from eachblock were placed on the Discovery™ in situ module and put throughprotocol Depar 10.

Slides are dry heated to 65° for 6 minutes then rinsed with 0.1M EDTAbuffer pH 8. After rinsing, the slide is incubated at 65° for 20minutes. Slides were then cooled to 37° C. and rinsed with APK Wash. Twoslides from each block were left untreated as controls. Following theDepar 10 treatment two treated slides from each block and one untreatedslide were stained for H & E as described in Example 1. Two treatedslides from each block and one untreated from each block are stained forLCA. Run outcomes: for both the H & E and antibody staining there was nostaining difference between the treated and untreated slides.

Example 8 Automated “Exposing” and “Cell Conditioning” forImmunohistochemistry

Various antibodies were assayed in the NexES Plus® (Ventana MedicalSystems, Inc., Tuscon, Ariz.) automated environment according to thefollowing protocol and flow chart:

Protocol:

-   -   (1) ***** Select EZ Prep *****    -   (2) ***** Start Timed Steps *****    -   (3) If deparaffinization is selected    -   (4) Warm slide to 75° C. and incubate for 4 minutes    -   (5) Adjust volume    -   (6) Apply LCS™    -   (7) Incubate for 8 minutes    -   (8) Rinse slide    -   (9) Adjust volume    -   (10) Apply LCS™    -   (11) Warm slide to 42° C. and incubate for 2 minutes    -   (12) If cell conditioning is selected    -   (13) If conditioner #1 is selected    -   (14) Rinse slide    -   (15) Adjust slide volume    -   (16) Apply LCS™    -   (17) Warm slide to 42° C. and incubate for 2 minutes    -   (18) Apply cell conditioning coverslip    -   (19) Warm slide to 100° C. and incubate for 2 minutes    -   (20) Apply cell conditioner #1    -   (21) Apply LCS™    -   (22) If standard is selected    -   (23) Incubate for 6 minutes    -   (24) Apply cell conditioner #1    -   (25) Apply LCS™    -   (26) Repeat (21)-(23) 9 times    -   (27) Warm slide to 42° C. and incubate for 2 minutes    -   (28) If mild is selected    -   (29) Incubate for 6 minutes    -   (30) Apply cell conditioner #1    -   (31) Apply LCS™    -   (32) Repeat (27)-(29) 4 times    -   (33) Warm slide to 42° C. and incubate for 2 minutes    -   (34) If extended is selected    -   (35) Incubate for 6 minutes    -   (36) Apply cell conditioner #1    -   (37) Apply LCS™    -   (38) Repeat (33)-(35) 14 times    -   (39) Warm slide to 42° C. and incubate for 2 minutes    -   (40) If conditioner #1 is not selected    -   (41) If conditioner #2 is selected    -   (42) Rinse slide    -   (43) Adjust slide volume    -   (44) Apply LCS™    -   (45) Warm slide to 42° C. and incubate for 2 minutes    -   (46) Apply cell conditioning coverslip    -   (47) Warm slide to 100° C. and incubate for 2 minutes    -   (48) Apply cell conditioner #2    -   (49) Apply LCS™    -   (50) If standard is selected    -   (51) Incubate for 6 minutes    -   (52) Apply cell conditioner #1    -   (53) Apply LCS™    -   (54) Repeat (49)-(51) 9 times    -   (55) Warm slide to 42° C. and incubate for 2 minutes    -   (56) If mild is selected    -   (57) Incubate for 6 minutes    -   (58) Apply cell conditioner #1    -   (59) Apply LCS™    -   (60) Repeat (55)-(57) 4 times    -   (61) Warm slide to 42° C. and incubate for 2 minutes    -   (62) If extended is selected    -   (63) Incubate for 6 minutes    -   (64) Apply cell conditioner #1    -   (65) Apply LCS™    -   (66) Repeat (61)-(63) 14 times    -   (67) Warm slide to 42° C. and incubate for 2 minutes    -   (68) Rinse slide    -   (69) Adjust slide volume    -   (70) Apply LCS™    -   (71) Disable slide heater    -   (72) ***** Select Reaction Buffer *****

Table I summarizes the results from automated immunohistochemistryassays performed on the NexES Plus (also known as the BENCHMARK™ IHC)using the automated deparaffinization and cell conditioning protocolsdescribed herein. The slides from the automated immunohistochemistryassays were compared to slides prepared using manualdeparaffinization/cell conditioning protocols on a NexES instrument.Specific antibody slides were prepared in triplicate. Negative controlslides were prepared singly. Slides from both assays were analyzed bypersons skilled in the art and assigned ratings for specific stainingintensity and non-specific background staining intensity. The ratingsfor all of the slides from the NexES Plus fully automated assaysexceeded the ratings for the manually deparaffinized and cellconditioned NexES slides in both specific staining intensity andnon-specific background intensity. Slides were analyzed and rated by aminimum of two independent persons skilled in the art and averagestaining intensities were determined. The column marked “QualifiedTissue” in Table 1 represents the standard control tissue used tostandardize the antibody used on it. The standardized antibody is givena rating of 4.0. As can be seen in the column marked “Average StainingIntensity,” most of the automated deparaffinized/cell conditioned slidesmet or exceeded those results. TABLE 1 Antibody and Cell ConditioningResults Incubation Average Qualified Time Negative Staining NegativeAntibody Clone Tissue Pretreatment (minutes) Control Intensity Controlanti-PR 1A6 Endo11299#2 60 min. cell conditioning 32 NC Ig 4 0 anti-ER6F11 UT31698V 60 min. cell conditioning 32 NC Ig 4 0 anti-Pan KeratinAE1, AE3, PCK26 SK197A  4 min. Protease 1 16 NC Ig 4 0 anti-C-erbB-2CB11 TMC197G 60 min. cell conditioning 32 NC Ig 4 0 anti-S100 polyclonalp19 None 16 Rbt NC 4 0 anti-CD20 L26 T1398H None 16 NC Ig 3.9 0anti-Vimentin 3B4 245A  8 min. Protease 2 16 NC Ig 4 0 anti-LCA RP2/18T31099B None 16 NC Ig 4 0 anti-Melanosome HMB45 97-715C None 16 NC Ig 40 anti-CEA TF-3H8-1 CCA98157 None 16 NC Ig 4 0 anti-CD45RO A6 T9810163None 16 NC Ig 3.6 0 anti-Chromogratin LK2H10 pN None 16 NC Ig 4 0anti-EMA Mc5 BRCA98139BC None 16 NC Ig 3.75 0 anti-Kappa PolyclonalT4282  4 min. Protease 1 16 Rbt NC 4 0 anti-Lambda Polyclonal 51099  4min. Protease 1 16 Rbt NC 4 0 anti-CD15 MMA K121698 None 16 NC Ig 4 0anti-Desmin DE-R-11 SI173BC 16 min Protease 2 16 NC Ig 4 0 anti-MuscleActin HUC1-1 DIA8699A None 16 NC Ig 4 0 anti-PSA Polyclonal 271A None 16Rbt NC 4 0 anti-Ki67 MM1 P92-3622 60 min. cell conditioning 16 NC Ig 3.90 anti-p53 bp-53-11 EV246B 60 min. cell conditioning 16 NC Ig 4 0anti-Keratin 5D3 L103CB  8 min Protease 1 16 NC Ig 4 0 anti-NSEBBS/NC/VI-H14 #2 None 16 NC Ig 3.9 0 anti-bcl-2 bcl-2/100/D5 Block6 60min. cell conditioning 32 NC Ig 4 0 anti-CD30 1G12 HodgkinsI912696C 60min. cell conditioning 16 NC Ig 3.75 0 anti-CD43 L60 T11698H None 16 NCIg 3.75 0 anti-GFAP Polyclonal CBR9872E None 16 Rbt NC 4 0anti-Synaptophysin 27G12 406A ND 16 ND 4 0NOTE:In Table 1, ND stands for No Data.

The primary antibodies (available from Ventana Medical Systems, Inc.,Tuscon, Ariz.) referred to in Table 1 (above) are described below, inalphabetical order:

-   -   anti-bcl-2:        -   Clone bcl-2/100/D5 is a monoclonal antibody used to detect            bcl-2, which is an inhibitor of apoptosis. This antibody may            be an aid to distinguish between reactive and neoplastic            follicular proliferation. Control: Tonsil. (VMSI # 760-2693)    -   anti-C-erbB-2 (HER2/neu):        -   Clone CB11 is a monoclonal antibody to c-erb-B2, which is            localized on the cell membrane, and occasionally, in the            cytoplasm of some neoplastic cells. (VMSI # 760-2994)    -   anti-CD15:        -   Clone MMA is a monoclonal antibody used to aid in the            identification of cells of the granulocytic lineage and/or            Reed-Sternberg differentiation. Control: Hodgkin's Lymphoma.            (VMSI # 760-2504)    -   anti-CD20:        -   Clone L26 is a monoclonal antibody used to aid in the            identification of cells of the B lymphocytic lineage.            Contol: Tonsil. (VMSI # 760-2531/760-2137)    -   anti-CD43:        -   Clone L60 is a monoclonal antibody that specifically binds            to antigens located in the plasma membrane of normal            granulocytes and T lymphocytes. Control: Tonsil. (VMSI #            760-2511)    -   anti-CD45RA:        -   Clone X148 is a monoclonal antibody that specifically binds            to antigens located in the plasma membrane of normal B            lymphocytes and a subset of T lymphocytes. Control: Tonsil.            (VMSI # 76-2510)    -   anti-CD45RO:        -   Clone A6 is a monoclonal antibody that binds to the plasma            membrane of cells of the T lineage and a subset of B-cells.            Control: Tonsil. (VMSI # 760-2563)    -   anti-CEA:        -   Clone TF-3H8-1 specifically binds to antigens located in the            plasma membrane and cytoplasmic regions of mucosal            epithelial cells. Control: Colon Carcinoma. (VMSI #            760-2507/760-2141)    -   anti-Chromogranin:        -   Clone LK2H10 is a monoclonal antibody that binds the            chromogranin protein located in the secretory granules of            normal and neoplastic neuroendocrine cells. Control:            Pancreas. (VMSI # 760-2519/760-2140)    -   anti-Desmin:        -   Clone DE-R-11 is a monoclonal antibody used to aid in the            identification of cells of the myocytic lineage. Desmin is            an intermediate filament found in mature smooth, striated            and cardiac muscle. Control: Vas Deferens. (VMSI # 760-2513)    -   anti-EGFR:        -   Clone 31G7 is directed against a transmembrane glycoprotein            present on a variety of cells. (VMSI # 760-2548)    -   anti-EMA:        -   Clone Mc5 is a monoclonal antibody used to aid in the            identification of cells of epithelial lineage. EMA is of            value in distinguishing both large-cell anaplastic carcinoma            from diffuse histiocytic lymphoma, and small-cell anaplastic            carcinoma from well and poorly differentiated lymphocytic            lymphomas. Control: Carcinoma. (VMSI # 760-2508)    -   anti-ER:        -   Clone 6F11 is a monoclonal antibody used to detect the            presence of estrogen receptor. (VMSI # 760-2596/760-2132)        -   Clone CC4-5 is a monoclonal antibody also used to detect the            presence of estrogen receptor. (VMSI #760-2546/760-2138)    -   anti-GFAP:        -   GFAP polyclonal antibody is directed against glial            fibrillary acidic protein present in the cytoplasm of most            human astrocytes and ependymal cells. This reagent may be            used to aid in the identification of cells of the glial            lineage. Control: Brain. (VMSI # 760-2516)    -   anti-Kappa:        -   Kappa light chains are expressed by cells of the B-cell            lineage. Light chain production by lymphoid cells is            genetically restricted such that the immunoglobulin            molecules produced by an individual cell will only contain a            single light chain class. This clonal restriction may be            used to indicate the polyclonal or monoclonal nature of            B-cell and plasma cell populations. Control: Plasmacytoma.            (VMSI # 760-2514)    -   anti-Keratin:        -   Clone 5D3 is a monoclonal antibody raised against human            epidermal keratins. This antibody may be used to aid in the            identification of cells of the epithelial lineage. This            antibody reacts with cytokeratins 8 and 18. Control:            Carcinoma. (VMSI # 760-2501)        -   Clone AE1 is a monoclonal antibody raised against human            epidermal keratins. This antibody may be used to aid in the            identification of cells of the ductal epithelial lineage.            AE1 reacts with cytokeratins 10, 13, 14, 15 and 19. Control:            Liver. (VMSI # 760-2521)    -   anti-Pan Keratin:        -   Pan Keratin is a cocktail of monoclonal antibody clones            AE1/AE3/PCK26 used to aid in the identification of cells of            the epithelial lineage. This antibody cocktail reacts with            all cytokeratins except 9, 11 and 12. Control: Skin. (VMSI #            760-2595/760-2135)    -   anti-Ki67:        -   Clone MM1 is a monoclonal antibody that specifically binds            to nuclear antigens associated with cell proliferation that            are present throughout the active cell cycle (G1, S, G2 and            M phases), but absent in resting (G0 phase) cells. This            reagent may be used to aid in the identification of            proliferating cells. Control: Tonsil. (VMSI # 760-2520)    -   anti-Lambda:        -   Lambda light chains are expressed by cells of the B-cell            lineage. Light chain production by lymphoid cells is            genetically restricted, such that the immunoglobulin            molecules produced by an individual cell will only contain a            single light chain class. This clonal restriction may be an            aid to indicate the polyclonal or monoclonal nature of            B-cell and plasma cell populations. Control: Plasmacytoma.            (VMSI # 760-2515)    -   anti-LCA:        -   Clone RP2/18 is a is a monoclonal antibody used to aid in            the identification of cells of lymphocytic descent. This            antibody specifically binds to antigens located            predominantly in the plasma membrane and cytoplasmic rim of            lymphocytes with variable reactivity to monocytes/histocytes            and polymorphs. Control: Lymphoma. (VMSI #            760-2505/760-2136)    -   anti-Melanosome:        -   Clone HMB45 monoclonal antibody is used to aid in the            identification of cells of the melanocytic lineage. It            reacts with an antigen expressed in abnormal melanocytes and            melanoma cells. Control: Melanoma. (VMSI # 760-2518M60-2139)    -   anti-Muscle Actin:        -   Clone HUC1-1 is a monoclonal antibody used to aid in the            identification of cells of myocytic descent. Muscle actin is            expressed in cells of the striated, smooth and cardiac            muscle lineage. Control: Ileum. (VMSI # 760-2502)    -   anti-NSE:        -   Clone BBS/NC/VI-H14 is a monoclonal antibody that reacts            with an antigen that may be expressed in the cytoplasm of            neurons, neuro-endocrine cells, endocrine tumors, carcinoids            and Merkel cell tumors. Control: Pancreas. (VMSI # 76-2517)    -   anti-p53:        -   Clone Bp-53-11 is a monoclonal antibody directed against the            p53 protein. This reagent may be used to aid in the            identification of abnormally proliferating cells in            neoplastic cell populations. Control: Carcinoma. (VMSI #            760-2540)    -   anti-PCNA:        -   Clone PC10 is a monoclonal antibody that may be used to aid            in the identification of proliferating cells in various cell            populations. The PCNA antigen is expressed in all            proliferating cells in the G1, S, G2 and M phases. Control:            Tonsil. (VMSI # 760-2503)    -   anti-PSA:        -   PSA polyclonal antibody reacts with the secretory protein            expressed in the cytoplasm of prostate epithelial cells.            Control: Prostate. (VMSI # 760-2506)    -   anti-PSAP:        -   Clone PASE/4LJ is a monoclonal antibody used to aid in the            identification of cells of the prostate lineage. PSAP            specifically binds to antigens located in the cytoplasmic            regions of the normal prostate epithelial cells. Control:            Prostate. (VMSI # 760-2509)    -   anti-PR:        -   Clone 1A6 is a monoclonal antibody used to aid in the            identification of the progesterone receptor in human tissue.            (VMSI # 760-2547/760-2133)    -   anti-S100:        -   S100 polyclonal antibody is used to aid in the            identification of cells of normal and abnormal            neuro-endocrine descent. Control: Skin. (VMSI #            760-2523/760-2133)    -   anti-Vimentin:        -   Clone 3B4 is used to aid in the identification of cells of            mesenchymal origin. Control: Vas Deferens. (VMSI #            760-2512/760-2134)    -   Negative Control:        -   Polyclonal serum applied to negative tissue controls as part            of quality control procedures for polyclonal antibodies.            (VMSI # 760-1023)    -   Negative Control Ig:        -   Clone MOPC-21 is applied to negative tissue controls as part            of quality control procedures for monoclonal antibodies.            (VMSI # 760-2014)

Example 9 Automated “Cell Conditioning” with or without “Exposing” forImmunohistochemistry (IHC), Immunocytochemistry (ICC) or In situHybribidization (ISH)

The CC2 solution may be used on automated immunohistochemical,immunocytochemical or nz situ hybridization instruments (e.g.,BenchMark™ ISH, Ventana Medical Systems, Inc., Tucson, Ariz.; BenchMark™IHC, Ventana Medical Systems, Inc., Tucson, Ariz.; etc.) with reagentsdesigned to detect the c-erb2/HER-2/neu gene or protein(s) expressedtherefrom (e.g., INFORM® HER-2/neu DNA test, Ventana Medical Systems,Inc., Tucson, Ariz.; PATHWAY™ HER-2/neu (CB11) protein test, VentanaMedical Systems, Inc., Tucson, Ariz.; etc.).

For an ISH assay, the CC2 solution may preferably be used in anautomated pretreatment step following deparaffinization with the CC2solution applied to the tissue at about 90° C. for about 10 minutesprior to application of the DNA probe. The heat treatment in conjunctionwith the CC2 solution allows the genomic DNA to become better suited forbinding with the c-erb2/HER-2/neu DNA probe.

For an IHC assay, the CC2 solution may preferably be used in anautomated cell conditioning step following deparaffinization with theCC2 solution applied to the tissue at about 95° C. for about 30 minutes.The high temperature in conjunction with the CC2 solution allows thec-erb2/HER-2/neu protein (antigen or epitope) to become better suitedfor binding with the anti-c-erb2/HER-2/neu antibody.

These are representative contexts in which the CC2 solution ispreferably used. The CC2 solution is also useful in many other assays,techniques, protocols or procedures designed to detect the presence orabsence of targeted. DNA or protein molecules under consideration.

From the foregoing detailed description, it will be appreciated thatnumerous changes and modifications can be made to the aspects of theinvention without departure from the true spirit and scope of theinvention. This true spirit and scope of the invention is defined by theappended claims, to be interpreted in light of the foregoingspecification.

1. An aqueous composition of matter, comprising citrate buffer, ethyleneglycol, sodium metabisulfite and a surfactant.
 2. A compositionaccording to claim 1, wherein the composition is buffered fromapproximately pH 4 to approximately pH
 9. 3. A composition according toclaim 1, wherein composition is buffered at approximately pH
 6. 4. Acomposition according to claim 1, wherein the composition comprises lessthan or equal to approximately 100 mM citrate buffer.
 5. A compositionaccording to claim 1, wherein the composition comprises approximately 10mM citrate buffer.
 6. A composition according to claim 1, wherein thecomposition comprises less than or equal to approximately 10% ethyleneglycol.
 7. A composition according to claim 1, wherein the compositioncomprises approximately 5% ethylene glycol.
 8. A composition accordingto claim 1, wherein the composition comprises less than or equal toapproximately 10 mM sodium metabisulfite.
 9. A composition according toclaim 1, wherein the composition comprises 1 mM sodium metabisulfite.10. A composition according to claim 1, wherein the compositioncomprises less than or equal to approximately 10% SDS.
 11. A compositionaccording to claim 1, wherein the composition comprises approximately0.3% SDS.
 12. A composition according to claim 1, wherein thecomposition comprises approximately 10 mM citrate buffer atapproximately pH 6, approximately 5% ethylene glycol, approximately 1 mMsodium metabisulfite and approximately 0.3% SDS.
 13. An aqueouscomposition of matter comprising a buffer, a glycol, sodiummetabisulfite and a surfactant.
 14. A composition according to claim 13,wherein the buffer is a citrate buffer.
 15. A composition according toclaim 14, wherein the buffer is at approximately pH
 6. 16. A compositionaccording to claim 13, wherein the glycol is ethylene glycol.
 17. Acomposition according to claim 13, wherein the concentration of thebuffer is less than or equal to approximately 100 mM, the concentrationof the glycol is less than or equal to approximately 10%, theconcentration of the sodium metabisulfite is less than or equal toapproximately 10 mM and the concentration of the SDS is less than orequal to approximately 10%.
 18. A composition according to claim 17,wherein the buffer is citrate at a concentration of approximately 10 mMand the pH of the composition is approximately
 6. 19. A compositionaccording to claim 18, wherein the glycol is ethylene glycol and theconcentration of ethylene glycol is approximately 5%, wherein theconcentration of sodium metabisulfite is approximately 1 mM and whereinthe concentration of SDS is approximately 0.3%.
 20. An aqueouscomposition of matter comprising approximately 10 mM citrate buffer atapproximately pH 6, approximately 5% ethylene glycol, approximately 1 mMsodium metabisulfite and approximately 0.3% surfactant.
 21. An aqueouscomposition of matter according to claim 20, wherein the surfactant isSDS.